Voltammetric estimation of residual nitroxynil in food products using carbon paste electrode

A simple and sensitive voltammetric method was developed and validated for the recognition of the veterinary drug nitroxynil (NTX). The method is based on studying its voltammetric behavior at a carbon paste electrode. Square wave voltammetry (SWV) was successfully applied in this study. The anodic peak current obtained was a linear function of NTX concentration in Britton Robinson buffer of pH 3 over the range of 3.9 × 10–6–1.0 × 10−4 M with lower detection and quantitation limits of 3.1 × 10–7 and 9.4 × 10–7 M, respectively. The proposed method was first applied to the assessment of the drug in commercial vials. The method was further used to monitor the residual amounts of the drug in bovine meat, kidney, fat, and milk samples. The results obtained were favourably compared with those given by reference method. The interference likely to be introduced by co-administered drugs was evaluated. The electrode reaction was elucidated, and electron transfer kinetics were studied.


Equipments.
Voltammetric measurements were carried out using a Metrohm Voltamograph (884 Professional VA, Utrecht, Netherlands). One compartment cell with a three-electrodes set-up including carbon paste electrode coupled with a reference electrode (Ag/AgCl/KCl (3 M) and Pt auxiliary electrode. The electrochemical process was performed at the ambient temperature of 25 °C. The stored results were retrieved using VIVA 2.1 software. The pH measurements were conducted and regulated by a pH Meter (Jenway 3505 Instruments, UK). A vortex mixer (Zx4, VELP ScientificaSrl, Italy) was used for blending the solutionsand centrifuge (Model Sigma 2-16P, Germany). A glass homogenizer (glass-col, 099ck424, Korea) was utilized for sample homogenization. (CPE). CPE was prepared by mixing 0.5 g of graphite powder and three drops (0.3 mL) of paraffin oil in a small agate mortar. The homogenized paste was introduced into an insulin syringe with a cross-section of 1.34 mm and flattened on a filter paper to obtain a polished appearance. For electrochemical measurements, a copper wire was touched with the carbon paste from the syringe end side and used to connect with the electrochemical device as a working electrode. After every measurement, the paste was neatly removed before pressing a new section into the electrode.

Procedures. Preparation of working electrode
Preparation of standard and working solutions. Electrochemical measurements. The stock solution of NTX (1.0 × 10 -3 M) was prepared by dissolving 14.0 mg of the pure drug in 50 mL of methanol in a measuring flask, then sonicating until complete dissolution. Various serial dilutions were performed to get different concentrations of NTX (1.0 × 10 −4 -1.0 × 10 −6 M) by completing aliquots of standard solution with BRb of pH 3. All prepared solutions were kept at 4 °C and protected from light. The standard solution is stable for at least 2 weeks.
Cyclic and square-wave voltammetric modes were utilized for the implementation of the electrochemical studies of NTX at the CPE surface. The optimization of SWV parameters was done at different values for potential step (3-20 mV), frequency , pulse amplitude (10-60 mV), and various scan rates (20-300 mV s −1 ). NTX monitoring was achieved by adopting the SWV mode at the following optimized parameters: a potential step of 20 mV, frequency of 20 Hz, pulse amplitude of 60 mV, and a scan rate of 100 mV s −1 . All scans were carried out in the positive direction with an applied potential range of + 20 to + 1600 mV at ambient temperature. The variation of pH over the range of 2.5-9 was applied to investigate its effect on the cyclic voltammetric behavior of the drug. Application to bovine meat, kidney, and fat samples. NTX was spiked to bovine meat, kidney, and fat samples. Each sample (5 g) was accurately weighed and homogenized with NTX and methanol at 5000 rpm for 5 min. The homogenate was sonicated for 15 min then centrifuged at 3000 rpm for another 5 min. 2 mL of the supernatant of all samples was transferred into 10 mL volumetric flasks and completed to the mark with methanol and filtered through 0.45 μm syringe filters. Then aliquots of NTX working standard solutions equivalent to (1.7 × 10 -6 -6.8 × 10 −4 M) were transferred into a series of 25 mL volumetric flasks and completed to the mark with BRb of pH 3. The linearity was investigated by plotting the peak current (Ip) versus drug amount (M).
Preparation of bovine milk samples. Milk sample (5 mL) was transferred into a 25 mL volumetric flask, spiked with aliquots of NTX standard solution then vortex mixed. Protein precipitation was carried out by adding 5 mL of 1 N HCL 39 , and the supernatant was filtered through 0.45 μm syringe filters and transferred to a 25 mL volumetric flask. The volume was completed to the mark with methanol. Aliquot of the previously filtered supernatant was then transferred to the voltammetric cell, completed to the mark with BRb of pH 3, and the SW voltammograms were recorded. Then the % recoveries were calculated.

Results and discussion
Electrochemical investigation of NTX at CPE. Effect of pH. Preliminary studies using cyclic voltammetry display the behavior of irreversible oxidation of NTX at CPE. Figure 2 demonstrates typical cyclic Voltammograms of 1.18 × 10 −5 M of NTX in BRb at different pHs over the range of 2.5-9, at a sweep rate of 100 mV/s at CPE. BRb was chosen as the supporting electrolyte in all measurements 24 . Figure 2A was plotted between peak potential Ep (V) against different pHs, and as shown, the peak potentials are shifted to less positive values by increasing the pH of the tested solutions. These results signify that the oxidation of NTX is pH-dependent, and it exhibited a linear regression plot obeying the following equation: www.nature.com/scientificreports/ Implying that protons and electrons are directly involved in the oxidation process. The highest anodic current for NTX was obtained at pH 3. Considerable lowering in Ip values with increasing pH values till pH 6 was noticed, but a significant increase at higher pHs was observed (Fig. 2B). Thus pH 3 was chosen as the most suitable one for further investigations.
Effect of scan rate. The electrochemical behavior of NTX was investigated using various scan rates (20-300 mV s −1 ) for 1.96 × 10 −5 M of NTX in BRb of pH 3. The electrochemical mechanism could be explained by plotting the relationship between peak current and scan rates as shown in Fig. 3. As the scan rate increased, the oxidation peak current increased; this process suggested the kinetics of redox reaction sites of NTX on CPE 40 . Diffusion or adsorption-controlled mechanisms were suggested on CPE in NTX determination 41 . Fig. 3A shows high linearity (r 2 = 0.994) of the relationship between the peak current (Ipa) and the square root of the scan rate (v 1/2 ) as expressed in Eq. (2); this specifies that the oxidation process was controlled by diffusion phenomenon 42 .
Furthermore, a linear relationship ((r 2 = 0.999) was exhibited from the plot of logarithm of peak current versus scan rates values (Fig. 3B) as seen in the following equation: The slope value (0.447) is close to 0.5, confirming that the proposed mechanism of the electrochemical process on CPE was controlled by diffusion of electroactive species. As shown in Fig. 3C, the electrochemical oxidation peak potential (Ep) also relied on scan rate values. As the sweep rates increased, the potentials were shifted to more positive values, and the derived Eq. (4) exhibited good linearity (r 2 = 0.994).
The kinetic parameters of the electron-transfer process were evaluated adopting Laviron's theory for the irreversible process, and exhibited the number of electrons relocated as seen in the following equation 43 : where, R is the gas constant (8.314 J K mol −1 ), T is the absolute temperature, F is the Faraday constant (96,485 Coulomb. mol −1 ), α is the electron transfer coefficient, and n is the number of the transferred electrons.  www.nature.com/scientificreports/ The slope from the linear relationship between potential against log scan rate was used to calculate αn. Using this method, the slope value is 0.113, from which αn value was calculated to be 0.523. As α was assumed to be 0.5 for all irreversible electron transfer via redox reactions, n was found to be 1.04, approximating referring to one electron transfer in the oxidation of NTX on CPE 44 .

Method validation. According to ICH guidelines 38 and The U.S. Food and Drug Administration (FDA)
recommendations and based on analytical procedures validation and documentations 45 , the proposed method was validated. The analytical behavior of the proposed electrochemical sensor for NTX was studied by analyzing three batches (3 replicates each) of the standard solutions to show the linear range, detection and quantification limits, and precision (standard deviation), accuracy (trueness).
Limits of detection and quantification and method linearity. LOD and LOQ were calculated to be 3.1 × 10 -7 and 9.4 × 10 -7 M, respectively, using to the following equations 38 : where "σ" is the standard deviation of intercept and "S" is the slope of the calibration curve. Figure 4 showed a wide linearity range of the method that exhibited over the range of 3.9 × 10 -6 -1.0 × 10 −4 M. Table 1 summarized the calibration data and the corresponding validation parameters.    Table 2 illustrates the analytical features of the reported ones and the proposed approach for NTX determination.
Accuracy and precision. To ensure the method's accuracy, triplicate analysis of three different concentrations were measured. The mean percentage recoveries were calculated as shown in Table 3. The proposed method's accuracy was statistically evaluated by comparing the results attained by the proposed method with those given by the official method 46 using the t-test and F-test. No significant difference was found between them. The official method depends on measuring the UV absorbance of an aqueous alkaline solution of the drug at 271 nm, both in pure form and injections.
Inter-day precision was assessed by measuring three different concentrations, in triplicates, in three consecutive assays. Nevertheless, intra-day precision was evaluated for three concentrations in triplicates at the same assay. The relative standard deviations were less than 2%, as shown in Table 4.   www.nature.com/scientificreports/ Method robustness. The robustness was tested by deliberated slight variations in the experimental conditions to exhibit unbiased results. The considered variables involved minor changes in pH (3.0 ± 0.4) and the applied time before each measurement (20 s ± 3 s). During the experimental procedure, these slight changes had no impact on the peak current strength, signifying the reliability of the applied method during the regular procedure.
Specificity. The specificity of the method was illustrated by exploring the effect of biological tissue matrices in bovine meat, kidney, milk, and fat samples as well as common excepients in veterinary formulations. Any of these did not affect the application of the suggested method for determination of NTX as revealed by the high percentage recoveries as illustrated in Tables 5 and 6.
Interference effect of commonly co-administered drugs. The potential interference likely to be introduced from commonly co-administered drugs was studied on the determination of NTX at 2.0 × 10 −5 M. A systematic study of interference caused by each of: mebendazole, albendazole, flubendazole, cefotaxime and ivermectin was carried out. The peak potential of NTX was at 1.25 V, while the peak potentials for mebendazole, albendazole, flubendazole, cefotaxime were 1.1, 1.05, 1.07, 0.925 V, respectively, and no peak at all in case of ivermectin. The analysis of the obtained responses, revealed that these co-administered drugs did not interfere with the proposed approach, being far from the peak potential of the drug.
Application to pharmaceutical preparation and food products. Pharmaceutical preparation. The proposed method was utilized successfully to assay NTX in its commercial vials, as shown in Fig. 5. The results were statistically compared with the official method using the t-test and F-test. The results were in good agreement with those obtained from the official method, as shown in Table 5.
Application to food samples. The determination of NTX residues in bovine meat, kidney, and fat samples was analyzed using the developed method. Before measurements, methanol was utilized for protein precipitation in all tissue samples.